In previous studies, we demonstrated the primary role of the polycationic polyamines in translational regulation by overexpression of a polyamine catabolic enzyme, spermidine/spermine acetyltransferase 1 (SAT1) in HEK293 cells. Upon rapid depletion of polyamines by transduction with the adeno SAT1 virus, protein synthesis was inhibited at the level of initiation, while no or little inhibition of synthesis of RNA or DNA occurred. In the current period, we have obtained evidence for enhanced protein synthesis in cells with increased cellular putrescine and spermidine, due to stabilization of ornithine decarboxylase (ODC) activity, the first step enzyme in polyamine biosynthesis. This finding resulted from a genome-wide siRNA screen that was designed to identify genes whose silencing would enhance recombinant protein production. Silencing of OAZ1, the gene that encodes the ODC antizyme 1, consistently increased production of recombinant proteins, including soluble, membrane-bound and secreted proteins. Silencing of OAZ1 increased the recombinant protein production at the translational level, not at the transcriptional level, underscoring the important role of polyamines in translational regulation. We also investigated the function of eIF5A by proteomic analyses of HeLa cells depleted of eIF5A by adeno-eIF5A shRNA transduction, to identify cellular pathways and proteins that are directly or indirectly influenced by eIF5A. We identified 3810, 1258 and 2750 unique proteins (with 2 unique peptides with >95% confidence level) in iTRAQ experiment 1, 2 and 3, respectively, and 972 proteins commonly detected in all three runs. Of these, 104 proteins with significantly altered levels (protein ratio 1.5 or 0.66, p value 0.05) at 72, and 96h of Ad-eIF5A-shRNA transduction were selected for further analyses for polyproline occurrence and functional ontology classification. No consistent relationship was observed between the content of polyproline motifs and the protein levels in eIF5A-depleted cells. Analyses of the functional ontology of the significantly altered proteins revealed specific biological processes that are prominently up-or down-regulated in eIF5A-depleted cells and identified protein folding as the major cellular process affected by the depletion of eIF5A. Activation of the unfolded protein response (UPR) was confirmed by increases in levels of UPR signaling molecules, especially p50-ATF6. Our data from these unbiased, quantitative, proteomic analyses demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells.